We have demonstrated that relatively pure cholinergic synaptosomes can be prepared from bovine superior cervical ganglia after a pretreatment of the preparation with collagenase. However, this collagenase treatment prevents the K ion-induced release of ACh from the preparation. This effect of collagenase appears to correlate with an inhibitory effect on neurostenin activity in the preparation. We will first determine if collagenase itself or some contaminant in the preparation is responsible for this effect. Subsequently, we will determine if the collagenase effect is referable to an effect on Ca2 ions transport or on neurostenin directly. We can now prepare synaptosomes without collagenase pretreatment and will utilize this preparation to (a) investigate if protein and ATP are released along with ACh; (b) determine the source of choline for ACh biosynthesis; and (c) determine the rates of uptake of choline and release of ACh. Finally we will attempt to develop a fluorescence histochemical procedure for ACh.